plasma membrane expression of heat shock protein 60 in ... · strepavidin-fitc, -pe, and -red670...

INFECTION AND IMMUNITY,0019-9567/99/$04.0010

Aug. 1999, p. 4191–4200 Vol. 67, No. 8

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Plasma Membrane Expression of Heat Shock Protein 60 InVivo in Response to Infection

CINDY BELLES, ALICIA KUHL,† RACHEL NOSHENY, AND SIMON R. CARDING*

Department of Clinical Studies, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6010

Received 4 February 1999/Returned for modification 29 March 1999/Accepted 16 April 1999

Heat shock protein 60 (hsp60) is constitutively expressed in the mitochondria of eukaryotic cells. However,it has been identified in other subcellular compartments in several disease states and in transformed cells, andit is an immunogenic molecule in various infectious and autoimmune diseases. To better understand thefactors that influence expression of hsp60 in normal cells in vivo, we analyzed its cellular and subcellulardistribution in mice infected with the intracellular bacterium Listeria monocytogenes. Western blotting ofsubcellular fractionated spleen cells showed that although endogenous hsp60 was restricted to the mitochon-dria in noninfected animals, it was associated with the plasma membrane as a result of infection. The low levelsof plasma membrane-associated hsp60 seen in the livers in noninfected animals subsequently increased duringinfection. Plasma membrane hsp60 expression did not correlate with bacterial growth, being most evidentduring or after bacterial clearance and persisting at 3 weeks postinfection. Using flow cytometry, we deter-mined that Mac-11, T-cell receptor gd1, and B2201 cells represented the major Hsp601 populations inspleens of infected mice. By contrast, B2201 cells were the predominant hsp601 population in livers of infectedmice. Of the immune cells analyzed, the kinetic profile of the gd T-cell response most closely matched that ofhsp60 expression in both the spleen and liver. Collectively, these findings show that during infection hsp60 canbe localized to the plasma membrane of viable cells, particularly antigen-presenting cells, providing a meansby which hsp60-reactive lymphocytes seen in various infectious disease and autoimmune disorders may begenerated and maintained.

Heat shock proteins (hsps) are highly conserved families ofproteins which play essential roles in the folding, unfolding,and transport of proteins within both prokaryotic and eukary-otic cells (reviewed in reference 15). Although hsps are up-regulated under a number of stress conditions, many are con-stitutively expressed in normal cells (28, 32, 33). The fact thatdeletion of hsp60 is lethal (14) emphasizes the essential rolethat this particular protein plays in protein assembly and cel-lular growth. hsp60 exists as a multimer composed of 7 or 14hsp60 molecules in a toroid formation (30, 48) in the cytoplasmof prokaryotes or in the mitochondria of eukaryotes (4, 23). Ithas been estimated that at least half of the soluble proteins ofEscherichia coli can form complexes with bacterial hsp60(GroEL) while they are in unfolded or partially folded states(48), and hsp60 is utilized in the folding pathway for suchenzymes as mouse dihydrofolate reductase (8, 30, 48) andribulose-biphosphate carboxylase of Rhodospirillum rubrum (48).

hsp60 has been shown to be an immunodominant antigen ofmycobacteria and other microorganisms (13, 26, 27). Mycobac-terial hsp60 has been used as an adjuvant (39) and as a carriermolecule for conjugated vaccines (2, 38) and has been identi-fied as an antigen for ab T cells in a rodent model of Myco-bacterium tuberculosis-induced arthritis (47). Additionally, el-evated anti-hsp60 antibodies have been detected in patientswith juvenile chronic arthritis, diabetes mellitus, and cysticfibrosis (9), implying that hsp60 of either autologous or patho-genic origins may be present extracellularly in these diseases.The high degree of homology (.60%) between bacterial and

mammalian forms of hsp60 has raised the possibility that hsp60could be a potential antigen for autoimmune responses, and linkmicrobial infection and autoimmunity (28). In addition to homol-ogy with microbial antigens, human hsp60 has sequence homol-ogy with a number of other autoantigens (26). Cross-reactivitybetween pathogenic hsp60s and other autologous proteins may,therefore, play a role in some autoimmune diseases.

Although the functional hsp60 complex does not normallyappear outside the mitochondria in eukaryotes (23), severalrecent studies have identified hsp60 in other subcellular com-partments under certain stress conditions or disease states. Instudies using immunogold and electron microscopy, hsp60 hasbeen identified in the secretory granules and plasma mem-branes of pancreatic cells of nonobese diabetic mice prior tothe onset clinical disease (6). Mammalian hsp60 has been iden-tified on the surface of cells within the lesions of brain tissuefrom patients with multiple sclerosis (MS) (44, 45), in sectionsof intestinal tissue from patients with inflammatory bowel dis-ease (37), and in in vitro-propagated primary (24) as well astransformed (17) cell lines.

The significance of any extramitochondrial hsp60 expressionby normal nontransformed cells in intact animals and the invivo conditions under which this pattern of expression occursare not known. The purpose of this study, therefore, was toinvestigate the expression of hsp60 in vivo in mice infected withthe intracellular bacterium Listeria monocytogenes to deter-mine whether hsp60 expression increases in various subcellularcompartments during the course of infection and the hostimmune response, and to identify any hsp60 surface-positive(hsp601) cells.

MATERIALS AND METHODS

Animals. Female BALB/cByJ mice were obtained from the Jackson Labora-tory (Bar Harbor, Maine) and were used between 8 and 12 weeks of age.

* Corresponding author. Mailing address: Department of ClinicalStudies, University of Pennsylvania School of Veterinary Medicine,3900 Delancy St., Philadelphia, PA 19104-6010. Phone: (215) 573-3022. Fax: (215) 573-6168. E-mail: [email protected].

† Present address: University of Colorado Health Science Center,Denver, CO 80262.

4191

on May 16, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

Antibodies and reagents. The hybridoma cell line 24G2, which secretes amonoclonal anti-mouse Fc receptor (FcR) antibody, was obtained from theAmerican Tissue Culture Collection (Rockville, Md.), and intact antibody wasisolated from culture supernatants by protein G affinity chromatography. Thefollowing antibodies were obtained from commercial sources. Anti-mouse T-cellreceptor (TcR) ab-fluorescein isothiocyanate (FITC) (clone H57-597), gd TcR-FITC/phycoerythrin (PE) (GL3), Mac-1-FITC (M1/70-15), F480-PE (F4/80),B220-FITC/PE (RA3-6B2), GR1-FITC (RB6-8C5), CD3-FITC (500-A2), CD3-biotin/PE (CT-CD3), anti-mouse immunoglobulin G1 (IgG1)-biotin (LO-MG1),anti-mouse IgG1-horseradish peroxidase (HRP) (LO-MG1), anti-rabbit-FITC,strepavidin-allophycocyanin, and strepavidin-alkaline phosphatase (AP) werepurchased from Caltag (San Francisco, Calif.); anti-mouse CD45.2-biotin (104)and Vd4-FITC (GL2) were obtained from Pharmingen (San Diego, Calif.);strepavidin-FITC, -PE, and -Red670 were obtained from Life Technologies(Gaithersburg, Md.); the anti-hsp60 antibody LK2 was purchased from Stress-Gen (Vancouver, British Columbia, Canada). The anti-TcRVd6.3 antibody 17Cwas generated in this laboratory (3). A rabbit anti-Listeria antiserum was kindlyprovided by Daniel Portnoy (University of California, Berkeley). A human an-tiserum reactive with subunits of the pyruvate dehydrogenase complex (PDC)from a patient with primary biliary cirrhosis was obtained from M. Eric Gershwin(University of California School of Medicine, Davis). Mouse and hamster IgGwas purchased from Sigma (St. Louis, Mo.).

Preparation of L. monocytogenes. Stocks of L. monocytogenes wild-type strain10403S were established as described previously (3). The LD50 (number ofbacteria which caused lethality in 50% of adult BALB/c mice injected intrave-nously with L. monocytogenes) was empirically determined to be 3 3 104 CFU.Mice were injected via the tail vein with L. monocytogenes diluted in 100 ml ofCa21- and Mg21-free) phosphate-buffered saline (PBS). The number of L.monocytogenes in the spleen and liver was determined by weighing a portion ofeach tissue, homogenizing the tissue in a Dounce homogenizer, plating serialdilutions of the homogenate on Luria broth agarose plates, and determining thenumber of cfu after 16 to 24 h of incubation at 37°C. Lysates of L. monocytogenesfor Western blotting experiments were prepared by lysozyme digestion andboiling as described elsewhere (41). Briefly, log-phase bacteria were pelleted,resuspended in 2 ml of lysozyme solution (2.5 mg/ml; Sigma), per ml, andincubated at 37°C for 90 min. The lysate was boiled for 5 min; 1 to 5 mg of proteinwas analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) to confirm cell lysis and protein integrity, aliquoted, and stored at270°C until used.

Cytofluorometric analysis. Four-color fluorescence was used for the kineticand phenotypic analysis of spleen and liver cells. Spleens and livers from infectedmice were minced into cold (4°C) Hanks-buffered saline with 7% fetal calf serumand antibiotics (Hfa medium for spleen cells or into Hepatozyme medium (LifeTechnologies) containing 5% fetal calf serum and antibiotics for liver cells.Tissues were passed through 100-gauge nylon mesh to obtain a single-cell sus-pension. One hundred-microliter aliquots of 106 cells were added to individualwells of V-bottom 96-well microtiter plates. Staining of liver cells was performedin Hepatozyme medium to preserve the membrane integrity of hepatocytes. Allantibody incubations were carried out on ice for 20 to 30 min each. Each samplewas first incubated with the monoclonal anti-mouse FcR antibody 24G2 andmouse and/or hamster IgG to reduce nonspecific reactivity of antibodies withFcR1 cells. After being washed with Hfa or Hepatozyme medium, cells wereincubated with biotin-conjugated antibodies, washed, and then incubated withfluorochrome-conjugated strepavidin and fluorochrome-conjugated antibodies.For controls, duplicate cell samples were incubated with isotype-matched anti-bodies of irrelevant specificity. Stained cells were run on a FACScan (BectonDickinson) and analyzed with CellQuest (Becton Dickinson) software.

Immunohistochemistry. A dual immunofluorescence-AP method was used tolocalize hsp601 and Mac-11 cells, or hsp601 and L. monocytogenes-infectedcells, in the spleens of mice after infection with a sublethal dose (0.36 to 0.38LD50) of L. monocytogenes. Spleens were removed intact and snap-frozen inTissue Tek (Miles Inc., Elkhart, Ind.)–2-methylbutane and stored at 280°C untilsectioned. Frozen 7-mm sections were fixed in acetone and incubated with mouseIgG (50 mg/ml) diluted in Tris-buffered saline (TBS; 10 mM Tris–150 mMNaCl)–1% bovine serum albumin (TBS-BSA) to prevent nonspecific binding ofantibodies. To visualize hsp601 and Mac-11 cells, sections were sequentiallyincubated with anti-Mac-1-biotin, strepavidin-AP, and anti-hsp60 (LK2)-FITC.To visualize hsp601 and L. monocytogenes-infected cells, sections were sequen-tially incubated with rabbit anti-L. monocytogenes antiserum, anti-hsp60 (LK2)-biotin, and anti-rabbit-FITC together with strepavidin-AP. Control, duplicatesamples were incubated with isotype-matched antibodies or normal serum. Theoptimal concentration of each antibody was empirically determined in a series ofpreliminary experiments. Antibodies were diluted in TBS-BSA, and all incuba-tions were performed in humidified chambers at 20°C for 30 min. In someinstances AP staining was amplified by using a tertiary rat AP anti-AP antibody(Serotec/Harlan Bioproducts, Indianapolis, Ind.). AP activity was visualized byusing the fluorescent Vector red substrate (Vector Laboratories, Burlingame,Calif.). Sections were counterstained with methyl green prior to mounting andphotomicroscopy.

Subcellular fractionation. All procedures for fractionation were performed at4°C. Cellular lysis and fractionation into nuclear, mitochondrial, cytoplasmic,plasma membrane, and secreted fractions were carried out by using a combined

and modified version of the methods of Jett et al. (25), Balch and Rothman (1),Depierre and Dallner (10), and Hubbard (22). Briefly, four to seven mice pertime point were sacrificed by cervical dislocation and immediately put on ice fordissection. Spleens and livers were removed, and a small portion of each takento determine L. monocytogenes CFU. The remainder was weighed, minced incold Dulbecco modified Eagle medium for spleens or cold Hepatozyme mediumcontaining 0.05% (wt/vol) collagenase (Sigma) for livers, passed through 100-gauge nylon mesh to separate cells, and centrifuged at 200 3 g for 5 min. Thesupernatant was collected as the secreted protein fraction. The cell pellet wasresuspended and washed in either Dulbecco modified Eagle medium or Hepa-tozyme medium, and the number and viability of cells were determined. Aliquotsof cell suspensions were also tested for the presence of succinate reductase (SR)to determine whether mitochondria were intact. If SR activity was detected, thenthe cell samples were discarded. Three million cells were removed for fluores-cence-activated cell sorting staining, and the remainder were lysed by the glycerolswell method (25). Glycerol in Hanks buffer (90%, vol/vol) was added to eachsolution in three equal increments 5 min apart for a final concentration of 30%glycerol. After the final addition of glycerol, cells were allowed to stand for 15min for osmotic swelling.

Cells were then centrifuged at 1,200 3 g for 10 min, and the supernatant wastaken as the glycerol fraction and tested for SR activity. Cells were then lysed bybeing quickly resuspending in hypotonic 0.25 M sucrose–10 mM Tris-HCl–1.5mM MgCl2 (0.25 M STM), incubated for 10 min with occasional vortexing, andthen broken by 10 strokes in a Dounce homogenizer. A small volume of thelysate was then taken for marker enzyme assays, including SR assay (11, 36, 46).Cell lysis was also evaluated microscopically. The lysate was vortexed and over-laid onto 800 ml of 2.5 M STM (2.5 M sucrose, 10 mM Tris-HCl, 1.5 mM MgCl2),which in turn was overlaid with 0.25 M STM prior to centrifugation at 100 3 gfor 50 min at 4°C. The cytosol (upper portion) and nuclear (lower portion)fractions were removed and assayed for marker enzyme activity and proteincontent. The membrane fraction (interface) was broken by repeated pipettingand vortexing for 1 min prior to further fractionation on a discontinuous sucrosegradient into fractions of 200 ml each at densities of 1.300 (2.5 M STM) to 1.00g/ml in increments of 0.015 g/ml (0.12 M STM) prepared in 5-ml polyvinylcentrifuge tubes. After centrifugation at 100 3 g for 75 min at 4°C, 13-dropfractions were collected by using a pump and capillary tubing until the first visiblemembrane interface was reached. Remaining fractions were removed by pipet-ting 250-ml increments from the gradient surface. Each fraction was then testedfor marker enzyme activity. Membrane fractions containing high levels of mito-chondrial marker enzyme (SR) activity and low levels of plasma membranemarker enzyme (alkaline phosphodiesterase I [APD]) activity were pooled as themitochondrial fraction. Fractions with high levels of APD and low levels of SRwere pooled and used as the plasma membrane fraction. All fractions wereassayed for protein content by using the Bio-Rad (Hercules, Calif.) protein assay,diluted or concentrated if necessary, and then immediately analyzed by Westernblotting. EL4 cell lysates were prepared by freezing intact cells in a dry ice-ethanol bath and transferring the cells to 220°C for 16 h, thawing them at roomtemperature, and boiling them for 5 min. The extent of cell lysis and proteinintegrity was evaluated by SDS-PAGE. Lysates were aliquoted and stored at270°C until used.

Marker enzyme assays. Colorimetric assays were used to determine the levelof enzyme activity in different subcellular fractions. Lactate dehydrogenase wasused as the marker enzyme for the cytosol and was assayed as described previ-ously (46). The assay for APD, used as the marker enzyme for the plasmamembrane, was done with sodium thymidine 59-monophosphate p-nitrophenylester (Sigma) as the substrate (36). The assay for SR, used as the marker enzymefor mitochondria, was done with 2-(p-indolphenyl)-3-(p-nitrophenyl-5-phe-nyl)tetrazolium (Sigma) as the substrate (36).

Western blotting. Fifteen micrograms of protein from each of the liver andspleen subcellular fractions or EL4 cell lysate and 20 mg of L. monocytogenescellular protein were electrophoretically separated on 5-10-15% step gradientSDS-polyacrylamide gel G and transferred to Hybond-C nitrocellulose (Amer-sham, Arlington Heights, Ill.). Membrane blots were first blocked with TBScontaining 5% dry milk powder for 60 min and then incubated with the mouseIgG1 anti-hsp60 antibody LK2 (80 mg/200 cm2), diluted in TBS–5% milk for 60min at 20°C. Blots were washed twice for 15 min in TBS with vigorous shakingand then incubated with 70 mg of anti-mouse IgG1-HRP in TBS–5% milk for 60min with slow shaking. The blots were then washed briefly twice (15 min each)and once for 14 to 16 h before development using an enhanced chemilumines-cence detection kit (Amersham). For reprobing of the blots with an anti-PDCantiserum, residual peroxidase activity was quenched, and the blots were incu-bated with the anti-PDC antiserum or normal human serum (1:4,000 dilution),washed, incubated with an HRP-conjugated anti-human Ig antibody (Caltag),and developed by chemiluminescence as described above.

RESULTS

Experimental approach. Female adult (6- to 8-week-old)BALB/c mice were infected intravenously (0.3 to 0.48 LD50)with of L. monocytogenes 10403S, and the two major sites ofinfection, liver and spleen, were analyzed for expression of

4192 BELLES ET AL. INFECT. IMMUN.


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

hsp60 by three independent methods. First, immunohisto-chemistry was used to identify hsp60 in situ in infected tissues.Second, subcellular fractionation and Western blotting wereused to determine the subcellular distribution of hsp60 inspleen and liver cells of infected mice. Third, antibody stainingand flow cytometry were used to identify and quantitate spleenand liver cells that might express surface hsp60 prior to andafter infection.

Expression of hsp60 in situ in the spleen after infection withL. monocytogenes. Dual-label immunohistochemical analysiswas used to localize hsp60 expression in situ in organ sectionsfrom infected mice. Tissues were snap-frozen, sectioned, andstained with antibodies for hsp60 (LK2-FITC) and Mac-1(Mac-1–biotin plus avidin-AP) or with hsp60 (LK2-biotin plusavidin-AP) and a rabbit anti-L. monocytogenes antiserum (plusFITC-conjugated anti-rabbit Ig). Due to the inherently highautofluorescence and nonspecific staining in liver sections frominfected mice, spleen sections were used for the majority ofthese experiments.

In sections of spleen from noninfected mice, it was notpossible to detect any staining with the anti-hsp60 antibodyLK2 above that of sections stained with control antibodies(data not shown). In infected animals, high levels of stainingwith LK2 were evident at day 3 in the spleen (Fig. 1), with the

FIG. 1. Detection of hsp60 expression in spleens of Listeria-infected mice.Spleens from BALB/c mice 3 (top) and 6 (bottom) days after infection with L.monocytogenes (0.38 LD50) were sectioned, fixed in acetone, and stained withantibodies specific for macrophages (Mac-1-biotin plus avidin-AP; red cells) and

FIG. 2. Separation of mitochondria and plasma membrane of spleen andliver cells. Cells were fractionated, and individual fractions were tested foractivity of marker enzymes for mitochondria (SR; ■) and plasma membraneAPD; E). Fractions containing highest levels of each enzyme (arrows labeled Mfor mitochondria and PM for plasma membrane) were combined and used as thefinal mitochondrial and plasma membrane fractions in Western blot experi-ments. Profiles are typical of those obtained in all experiments.

hsp60 (LK2-FITC; light green cells). The bottom photomicrograph shows thatthe majority of Mac-11 cells are also hsp601 and appear yellow-orange. Thestaining patterns shown are typical of those obtained in more than six indepen-dent experiments.

VOL. 67, 1999 hsp60 EXPRESSION IN VIVO DURING INFECTION 4193


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

number of hsp60-stained cells increasing by 6 days postinfec-tion. The halo-like pattern of staining and absence of anypunctate cytoplasmic (mitochondrial) staining of intact cells innonpermeabilized tissue sections was consistent with surfacestaining by the anti-hsp60 antibody (Fig. 1). Dual-staining ex-periments demonstrated that some hsp601 cells also expressedMac-1, indicating that monocytes/macrophages were surfacehsp60 positive after infection. Of note, the presence of orange-stained (Vector red plus FITC) cells in the spleen of mice at 6days postinfection indicated that a large number of Mac-11

cells coexpressed hsp60.Since bacterial numbers peak between 3 and 5 days postin-

jection in both liver and spleen (see Fig. 6F and 7F), theincrease in level of hsp60 immunofluorescence staining seen at6 days postinfection did not correlate with the level of L.monocytogenes found in each organ. Additional immunohisto-chemical staining experiments using a rabbit anti-L. monocy-togenes antiserum in conjunction with the anti-hsp60 antibodydemonstrated that the majority of L. monocytogenes-infectedcells were not hsp60 surface positive (data not shown). Thisdoes not, however, exclude the possibility that at least some ofthe hsp601 macrophages that did not stain with the anti-L.monocytogenes antibody had previously taken up and destroyedthe bacteria.

Increased hsp60 in mitochondrial and plasma membranefractions of cells from livers and spleens of infected animals.Since the pattern of immunohistochemical staining was consis-tent with increased hsp60 in the plasma membrane of cellsfrom infected tissues, we investigated the subcellular distribu-tion of hsp60 in liver and spleen cells at various times afterinfection. A continuous subcellular fractionation protocol was

developed to separate the nuclear, mitochondrial, cytosolic,and plasma membrane fractions of spleen and liver cells.Equivalent amounts of total protein of each fraction were thenelectrophoretically separated, transferred to nitrocellulose,and probed for hsp60 by Western blotting. Since our maininterest was in evaluating the amount of hsp60 associated withthe plasma membrane and to minimize the possibility thathsp60 from mitochondria could contaminate other fractions,the subcellular fractionation protocol was developed to opti-mize the purity of the plasma membrane fraction.

Figure 2 depicts results that are typical of the final step inseparating the mitochondria from the plasma membrane andthe assaying of individual fractions from the discontinuousdensity gradient for the mitochondrial enzyme marker SR andthe plasma membrane enzyme marker APD. Fractions show-ing peak activity and minimal cross-contamination were col-lected, pooled, and used as the mitochondrial and plasmamembrane fractions in subsequent Western blotting experi-ments. In addition, nuclear and cytosolic fractions were alsotested for APD and SR activity to determine fraction purity(Table 1). In the liver, the level of contamination of the plasmamembrane fractions with SR ranged from 3.3% 6 2.1% of thetotal activity detected in all of the subcellular fractions on day6 to 16.6% 6 11.4% on day 1. The level of SR activity presentin the plasma membrane fractions of the spleen ranged from10.1% 6 1.4% on day 3 to 12.6% 6 0.1% on day 1. Asexpected, the highest levels of SR activity were found in themitochondrial fractions of both the liver (78 to 93%) andspleen (66 to 83%).

Equivalent amounts of total protein from each of the foursubcellular fractions of spleen and liver cells from mice at

TABLE 1. Determination of subcellular fraction purity by mitochondrial (SR) and plasma membrane (APD) marker enzyme activity

Day Fraction

% of total enzyme activity (mean 6 SD [n 5 3])

Liver Spleen

SR APD SR APD

0 Nuclear 2.4 6 1.9 2.8 6 2.5 11.4 6 1.0 4.2 6 0.1Mitochondrial 92.5 6 8.6 18.7 6 15.6 66.9 6 0.9 35.4 6 3.8Cytosolic 0.1 6 0.06 6.5 6 5.2 11.4 6 1.8 6.8 6 5.7Plasma membrane 4.9 6 6.6 76.2 6 16.1 10.3 6 1.7 53.6 6 1.9








http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

various times after infection were analyzed by Western blottingfor expression of hsp60, using antibody LK2 and chemilumi-nescence to detect bound antibody. Scanning densitometry ofdeveloped blots was used to determine the relative amountsand distribution of hsp60 in the fractions of each sample (Ta-ble 2). As controls, total cell lysates from L. monocytogenes andEL4, a mouse lymphoma cell line, were included on each gel(Fig. 3). Since the LK2 antibody reacted with EL4 but not L.

monocytogenes, the hsp60 identified on these blots was autol-ogous murine hsp60.

Endogenous hsp60 expression in the various subcellularcompartments changed during the course of infection (Fig. 3and Table 2) and remained high as long as 3 weeks afterinfection. In the liver, hsp60 expression in naive animals (day0) was mainly restricted to the mitochondrial fraction, al-though the cytoplasmic and plasma membrane fractions con-tained low levels of hsp60. During the course of infection, thelevel of hsp60 in the plasma membrane fractions increaseduntil day 6 and then declined by day 9. Levels of mitochondrialhsp60 also increased, peaking on day 3. A large increase inplasma membrane hsp60 was seen on day 26, more than 2weeks after resolution of the infection, and was over sixfoldhigher than the level of hsp60 seen in the mitochondria at thistime (Table 2).

In spleens of naive mice, hsp60 was, as expected, localized tothe mitochondria, no hsp60 was detected in the other cellularfractions, including the plasma membrane. Three days afterinfection, hsp60 was detected in the plasma membrane frac-tions, with relatively higher levels seen on day 6 (Fig. 3). Sub-sequently, the plasma membrane levels decreased at day 9 andthen increased again at 3 weeks postinfection, similar to thepattern seen in the liver. Consistent with the results of theimmunohistochemical studies, the profile of hsp60 expressionin the plasma membrane fractions did not coincide with that ofbacterial CFU in either organ (see Fig. 6F and 7F).

The purity of the plasma membrane fractions (Table 1) andlevel of hsp60 expression in each of the subcellular fractions(Table 2) strongly suggest that the plasma membrane-associ-ated hsp60 seen in organs from infected mice is unlikely to beattributable to contamination by mitochondrial proteins. If theplasma membrane-associated hsp60 was due to mitochondrialeakage or damage and cross-contamination, the plasma mem-brane fractions should contain similar amounts of other mito-chondrial proteins, such as SR. This is not the case. For exam-ple, in liver cell samples from day 6, at which high levels ofhsp60 are detected in plasma membrane fractions, approxi-mately one-third of the total hsp60 present is in the plasmamembrane fraction, whereas only 3% of the total mitochondriaSR activity is found in this fraction. Similarly, the day 6 plasmamembrane fraction in the spleen accounts for approximately20% of the total hsp60 but only 10% of the SR activity. Sam-ples from day 26 postinfection emphasize this point further,with 63% of the total hsp60 expression and 14% SR activity

TABLE 2. Subcellular distribution of hsp60 in spleen and liver fractions of Listeria-infected mice, determined by densitometry readings

Fraction% of total hsp60 expression (mean 6 SD [n 5 3])

Day 0 Day 1 Day 3 Day 6 Day 9 Day 26

LiverNuclear 2.1 6 4.0 22.0 6 4.7 11.5 6 5.9 13.1 6 7.5 18.4 6 7.5 14.0 6 1.4Mitochondrial 47.2 6 13.9 37.0 6 19.0 37.2 6 12.4 34.1 6 6.9 40.3 6 21.4 10.4 6 0.1Cytoplasmic 24.1 6 9.6 26.7 6 19.8 39.4 6 3.8 21.4 6 5.3 16.9 6 5.0 13.1 6 6.3PMa 26.7 6 9.8 14.3 6 5.5 11.8 6 4.4 31.5 6 3.4 24.4 6 18.4 62.5 6 7.9

PM as % of mitochondria 55.6 38.6 31.7 92.4 60.5 600.9

SpleenNuclear 6.2 6 3.4 2.0 6 2.0 15.2 6 14.3 18.3 6 11.9 9.2 6 8.4 0.1 6 0.5Mitochondrial 82.5 6 0.6 94.3 6 21.3 67.2 6 15.9 46.4 6 5.1 66.7 6 12.2 65.1 6 0.2Cytoplasmic 0.7 6 2.8 2.1 6 4.0 2.1 6 2.7 15.4 6 2.8 2.6 6 0.1 0.1 6 4.9PM 10.5 6 0.1 1.5 6 2.9 15.5 6 1.3 19.9 6 8.0 21.5 6 10.9 34.7 6 4.1

PM as % of mitochondria 12.7 1.6 23.1 42.9 32.2 53.1

a PM, plasma membrane.

FIG. 3. Subcellular distribution of hsp60 in vivo during infection. Spleen andliver cells from mice at various times (days [d]) after infection with L. monocy-togenes (0.34 LD50) were fractionated, and 15 mg of protein from the nuclear (N),mitochondrial (M), cytoplasm (C), and plasma membrane (P) fractions wereanalyzed for hsp60 expression by Western blotting. Molecular weight markers(std) and total cell lysates of L. monocytogenes (20 mg; L) and EL4 (15 mg; E)were included on each gel. The results shown are representative of those ob-tained in three independent experiments. The horizontal lines on each blotrepresent positions of molecular weight standards (97.4, 66, and 46 kDa).



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

present in the liver plasma membrane fraction, while 35% ofthe total hsp60 and 12% of the SR are present in the plasmamembrane fraction of the spleen. The subcellular redistribu-tion and localization to the plasma membrane of mitochondrialproteins was, however, not unique to hsp60.

Reprobing of the Western blots analyzed for hsp60 with anantiserum containing high-titer antibodies to the mitochon-drial PDC showed that subunits of this complex, particularlyE3, were also associated with the plasma membrane fractionsof spleen and liver cells (Fig. 4). Importantly, however, theprofiles of PDC and hsp60 plasma membrane expression werenonoverlapping. Plasma membrane-associated hsp60 was de-tected in the absence of PDC (compare data for day 3 in Fig.3 and 4) and vice versa (day 1 in Fig. 3 and 4). Plasma mem-brane expression of PDC was not consistent, and E3 expressionin the plasma membrane was sometimes higher than in mito-chondrial fractions, which argues against the redistribution ofPDC (or hsp60) being attributable to contamination by mito-chondrial proteins during the fractionation procedure.

Identification of cells expressing surface hsp60. Four-colorflow cytometric analysis was used to identify and quantitatecells of different lineages that express surface hsp60 before andat various times after infection. Light scatter properties and/orvital dye stains were used to exclude apoptotic and dead cellsfrom this analysis. The number of granulocytes (GR11),monocytes/macrophages (Mac-11), B cells (B2201), ab T cells

(abTcR1), and gd T cells (gdTcR1) was determined from thefrequency of stained cells and the total number of viable cellsrecovered from each tissue. Isotype-matched antibodies of ir-relevant specificity were used to determine levels of back-ground staining.

Representative anti-hsp60 antibody staining profiles forMac-11 spleen and liver cells are shown in Fig. 5. Consistentwith the results of the subcellular fractionation and Westernblotting experiments, flow cytometric analysis confirmedplasma membrane expression of hsp60. The profiles of hsp60expression in both the liver and spleen as determined by thesetwo methods were also comparable. In the liver, the totalnumber of hsp601 cells increased up to day 6 postinfection,after which it decreased at day 9 and then increased again atday 21 (Fig. 6F). As was seen in the immunohistochemistry andsubcellular fractionation experiments, hsp60 expression didnot correlate with bacterial numbers, which peaked on day 3and were no longer detected by day 9 postinfection (Fig. 6).

Macrophages, B cells, and gd T cells were the major hsp60-expressing cells in the liver. On day 6, the first peak of hsp601

cells, macrophages (31%), and gd T cells (27%) made up themajority of the hsp601 cells (Table 3). By day 21 postinfection,corresponding to a second peak in hsp601 cells, the majority ofhsp601 cells were B2201 (31%) or gd T cells (35%). Of note,the kinetic profile of gd T cells most closely matched that ofhsp601 cells, with the number of gd T cells peaking on days 6and 21 postinfection coincident with peaks of hsp601 cells(Fig. 6). As seen previously (3), Vd41 and Vd6.31 cells dom-inated the gd T-cell response to L. monocytogenes infection(data not shown). The largest number of gd T cells was seen at

FIG. 4. Subcellular distribution of the PDC in spleens and livers of Listeria-infected mice. Western blots originally probed with anti-hsp60 antibody (Fig. 3)were stripped and reprobed with an antiserum from a patient with primary biliarycirrhosis containing high-titer antibodies to PDC. Bound antibody was visualizedby chemiluminescence as described in the legend to Fig. 3. The subunits of PDCpresent in the samples are indicated on the left. The horizontal lines on each blotrepresent positions of molecular weight standards (97.4, 66, 46, and 30 kDa). d1,d3, and d6, 1, 3, and 6 days postinfection.

FIG 5. Increased hsp60 expression on macrophages in livers and spleens ofinfected mice. Spleen and liver cells obtained from mice at various times afterinfection were analyzed by flow cytometry. hsp60 expression by monocytes/macrophages was determined by gating on Mac-11 cells and analyzing for hsp60expression. The filled and open plots show profiles of staining obtained withanti-hsp60 (filled profiles) and an isotype-matched antibody, respectively; num-bers represent percentages of Mac-11 cells which are hsp601.



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

3 days postinfection and 21 days postinfection, at which timethe majority were also hsp601 (Fig. 6). Also of note was thelarge number of B2201 cells present in the liver at 21 dayspostinfection. By contrast, GR11 and abTcR1 cells reachedmaximal numbers on day 9. With the exception of abTcR1

cells at 9 days postinfection, ab T cells and granulocytes com-prised less than 6% of hsp601 cells throughout infection (Ta-ble 3).

As seen in the liver, the kinetic profile of hsp60 cell surfaceexpression in the spleen did not correlate with that of bacterialnumbers (Fig. 7F). Although the number of GR11, abTcR1,and gdTcR1 cells showed similar kinetic profiles in the spleenas in the liver, the profile of macrophages and B cells weredistinct (Fig. 7). In contrast to the liver, B2201 cells repre-sented the largest population of hsp601 cells in the spleen atall of the time points analyzed (Table 3). Similar to the liver,the kinetic profile of total gd T cells most closely matched thatof hsp601 cells (Fig. 7), and the response was dominated byVd41 or Vd6.31 cells (data not shown). In addition, gd T cellsalso made up a significant proportion (15 to 30%) of hsp601

cells, particularly after 3 days postinfection, similar to that seenin the liver (Table 3).

In summary, the profile of hsp60 expression in the livers andspleens of infected mice is concordant with that obtained bysubcellular fractionation and Western blotting. The composi-tions of hsp601 cells are, however, different in these tissues,although antigen-presenting (APCs) cells are the predominanthsp601 population in the liver (macrophages) and spleen (Bcells). Finally, whereas the kinetic profile of plasma mem-brane-associated hsp60 expression does not overlap with bac-terial numbers, it does parallel the kinetics of gd T-cell involve-ment in both liver and spleen.

DISCUSSION

The results described in this study demonstrate that thecellular and subcellular distribution of hsp60 in cells of theliver and the spleen changes in response to L. monocytogenesinfection. Using three independent experimental approaches,we have shown that although hsp60 is not found on cell sur-faces in healthy noninfected animals, L. monocytogenes infec-tion results in expression of hsp60 on the plasma membrane ofviable intact cells in the liver and spleen. In particular, APCssuch as B cells and macrophages are surface hsp601. In addi-

FIG. 6. Kinetic profiles of cellular response, hsp60 expression, and bacteria in the liver. Before and at various times after infection with L. monocytogenes (0.30LD50), the numbers of granulocytes (GR1), monocytes/macrophages (Mac-1), B cells (B220), ab T cells (abTcR) and gd T cells (gdTcR) present in the liver weredetermined from total cell counts, and the proportion of cells reactive with lineage-specific antibodies as determined by flow cytometry. Closed symbols, total cellnumbers; open symbols, numbers of hsp601 cells of each cell subtype. Panel F shows the bacterial CFU (E) and total number of surface hsp601 cells (✙). All valuesare means from four animals. Standard deviations were ,20% in all cases and for the sake of clarity are not shown.

TABLE 3. Distribution of hsp601 cells in the liver and spleen, determined by flow cytometric analysis

Day

% of total hsp601 cells (mean 6 SD [n 5 4])

Liver Spleen

B2201 Mac-11 abTCR1 gdTCR1 GR11 Other B2201 Mac-11 abTCR1 gdTCR1 GR11

0 8.8 6 2.8 29.7 6 11.6 5.8 6 2.4 15.4 6 7.3 0.9 6 0.8 39.3 6 11.0 65.6 6 21.3 15.4 6 5.2 4.1 6 3.7 15.0 6 7.4 0.0 6 0.01 17.0 6 5.6 49.0 6 14.9 2.9 6 1.7 25.9 6 13.6 1.1 6 0.3 4.2 6 2.7 57.7 6 16.5 20.4 6 10.6 3.3 6 3.0 14.6 6 5.9 4.5 6 1.43 7.6 6 3.3 27.3 6 9.3 2.0 6 1.4 43.1 6 15.3 0.8 6 0.4 9.2 6 3.0 48.8 6 22.9 22.5 6 9.4 1.1 6 1.0 24.9 6 8.3 2.7 6 1.26 15.9 6 2.8 31.5 6 18.8 5.1 6 2.8 27.5 6 4.5 1.4 6 0.7 18.5 6 2.8 45.8 6 7.0 13.1 6 5.2 9.0 6 3.2 30.1 6 4.3 1.5 6 0.49 5.5 6 3.2 17.1 6 7.1 15.8 6 6.2 48.7 6 17.1 1.9 6 1.1 11.1 6 3.2 58.7 6 21.6 9.9 6 1.8 8.3 6 3.5 17.0 6 1.3 3.2 6 1.621 31.1 6 4.3 7.1 6 4.7 1.5 6 0.8 35.0 6 12.8 0.9 6 0.8 24.4 6 6.4 54.5 6 9.7 11.1 6 1.9 6.6 6 2.4 23.7 6 1.4 0.5 6 0.4



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

tion, our results demonstrate that surface hsp60 expressiondoes not correlate with the kinetics of bacterial growth. Sur-prisingly, elevated levels of hsp60 are still present 2 weeks afterresolution of the disease.

Although we know neither how the subcellular distributionof hsp60 is regulated nor the biological significance of plasmamembrane-associated hsp60, there are several possible expla-nations. Increased hsp60 on cell surfaces could simply be anindicator of cellular stress, possibly produced directly by infec-tion or indirectly by cytokines and fever. Production and repairof proteins increase during stress and infection, and hsp60 isnecessary to these processes (15). hsps also have long half-lives, and so it is possible that hsp60 is transported to the cellsurface as a way to eliminate excess hsp. If hsp60 is expelledfrom the cell, hsp60-derived peptides would then become dis-played on the surfaces of professional APCs. Considering thathsp60 is a major immunogen of many bacterial pathogens (26,27) and that there is a high level of homology between bacterialand mammalian hsp60 (28), this process could sustain acti-vated T cells primed to respond to a number of pathogens, aswell as contribute to autoimmune disease.

Another possibility is that surface hsp60 acts as a signal ofstressed, activated, or damaged cells, targeting them for clear-ance by cytotoxic cells or macrophages as part of the tissuerepair process. In our infectious disease model hsp60 could beexpressed on preapoptotic or apoptotic cells. Increased levelsof hsp60 expression have been detected on membranes of cellsinduced to undergo apoptosis by treatment with dexametha-sone (40) and also in apoptotic cells isolated directly fromAIDS patients (40). T cells undergoing antigen-triggered apo-ptosis in vivo disappear from the lymph nodes and spleen andaccumulate in the liver, where they undergo apoptosis and arecleared from the body (20, 21). Livers of L. monocytogenes-infected mice contains a large population of small viable cells(as indicated by low forward scatter) that are highly granular(high side scatter) and increase in number up to day 21, at

which time 85% are B2201, 15% are gdTcR1, and 95 to 98%are hsp601 (2a). These cells may have already encounteredantigen and be undergoing activation-induced apoptosis, accu-mulating in the liver awaiting clearance.

Although examples of secretion of hsp60 have been de-scribed for parasites (13), we have been unable to detect se-cretion of hsp60 by spleen or liver cells in vivo during listeri-osis. We have also been unable to detect hsp60 in thesupernatants of cultured hybridoma lines expressing hsp60 ontheir cell surfaces (2a). It is possible that autologous hsp60exists at the cell surface as an intact toroid form rather than aspeptide fragments associated with major histocompatibilitycomplex molecules. Since the hsp60 toroid form often containsother proteins, and the hsp60 complex can retain denaturedproteins that it cannot fold (7, 15, 16), one possibility is thatunder conditions of hyperactivation or stress, hsp60 chaper-ones (abnormal) proteins to the cell surface for their elimina-tion. The observation that hsp60 in the plasma membrane ofthe human T-cell leukemic cell line CEM-SS attaches to theH2B histone protein and is released as a result of proteinkinase A-catalyzed phosphorylation (29) is consistent with theability of hsp60 to expel proteins from the plasma membraneunder certain conditions. Plasma membrane hsp60 might berecognized by T cells as a complex containing proteins the cellis trying to expel. It is also possible that the structural changesof the hsp60 toroid form that occur when it complexes withother cellular proteins (43) reveal conformational determi-nants recognized by immune cells.

It is interesting that of the cell populations analyzed duringL. monocytogenes infection, the kinetics of the gd T-cell re-sponse most closely matches that of hsp60 expression. Severalgroups have postulated that hsp60 is a ligand for gd T cells (5,35). gd T cells and hsp601 oligodendrocytes have been colo-calized in sections of demyelinated brain tissue from patientswith MS (44, 45), and gd T cells have been shown in vitro to killoligodendrocytes isolated from MS patients (12). Also, hsp60-

FIG. 7. Kinetic profiles of the cellular response, hsp60 expression, and bacteria in the spleen. Spleen cells were analyzed and the different populations wereenumerated as described in the legend to Fig. 6. Closed symbols, total cell numbers; open symbols, numbers of hsp601 cells of each cell subtype. Panel F shows bacterialCFU (E) and total number of surface hsp601 cells (✙). All values are means from four animals. Standard deviations were ,20% in all cases and for the sake of clarityare not shown.



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

reactive gd T cells have been isolated from the synovial fluid ofpatients with rheumatoid arthritis (19), and synovial gd T cellsfrom patients with Lyme arthritis can induce apoptosis of sy-novial CD41 ab T lymphocytes via Fas-Fas ligand interactions(49). hsp601 cells (37) and large numbers of mycobacterialhsp60-reactive gd T cells (31) are found in the intestinal lesionsof patients with inflammatory bowel disease. Additionally,populations of peritoneal exudate cells from L. monocytogenes-infected mice that were enriched for gd T cells have beenshown to proliferate and release gamma interferon after cul-ture with recombinant mycobacterial hsp60 (18).

We have previously shown that a large proportion of gd Tcells that accumulate in both livers and spleens of L. monocy-togenes-infected mice express a TcR encoded by either Vd6 orVd4 (3), both of which have been previously shown to correlatewith hsp60 reactivity (34). The observation that Vd61 gd Tcells from L. monocytogenes-infected mice reacts with plasmamembrane preparations of L. monocytogenes-elicited, hsp601

peritoneal exudate cells (36a) suggests that gd T cells mayindeed respond to and recognize hsp60 expressed in theplasma membrane of cells in response to infection. If these gdT cells react with hsp60 on the cell surface, they may recognizethe hsp60 alone or complexed with an interior protein in amanner similar to major histocompatibility complex presenta-tion of peptides. Although liver and spleen gd T cells havelimited g- and d-chain pairing, these receptors, particularly thed chain, have diverse junctional sequences (34, 42). These cellsmay, therefore, recognize the hsp60 portion of such a complexwith the g/d constant regions and any variation of interiorproteins with junctional regions. The molecular nature of gd Tcell-hsp60 interactions is currently being investigated in ourlaboratory.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grants AI-31972,AI-45993, and HL-51749 from the National Institutes of Health and bya grant from the University of Pennsylvania Research Foundation.

REFERENCES

1. Balch, W. E., and J. E. Rothman. 1985. Characterization of protein transportbetween successive compartments of the Golgi apparatus: asymmetric prop-erties of donor and acceptor activities in a cell-free system. Arch. Biochem.Biophys. 240:413–425.

2. Barrios, C., A. R. Lussow, J. van Embden, R. van der Zee, R. Rappuoli, P.Constantino, J. A. Louis, P.-H. Lambert, and G. Del Giudice. 1992. Myco-bacterial heat-shock proteins as carrier molecules II: the use of the 70-kDamycobacterial heat-shock protein as carrier for conjugated vaccines cancircumvent the need for adjuvants and Bacillus Calmette Guerin priming.Eur. J. Immunol. 22:1365–1372.

2a.Belles, C., and S. R. Carding. Unpublished observations.3. Belles, C., A. L. Kuhl, A. J. Donoghue, Y. Sano, R. L. O’Brien, W. Born, K.

Bottomly, and S. R. Carding. 1996. Bias in the gd cell response to Listeriamonocytogenes: Vd6.31 cells are a major component of the gd T cell re-sponse to Listeria monocytogenes. J. Immunol. 156:4280–4289.

4. Boog, C. J. P., E. R. de Graeff-Meeder, M. A. Lucassen, R. van der Zee,M. M. Voorhorst-Ogink, J. S. van Kooten, H. J. Geuze, and W. van Eden.1992. Two monoclonal antibodies generated against human HSP60 showreactivity with synovial membranes of patients with juvenile chronic arthritis.J. Exp. Med. 175:1805–1810.

5. Born, W. K., R. L. O’Brien, and R. L. Modlin. 1991. Antigen specificity of gdT lymphocytes. FASEB J. 5:2699–2705.

6. Brudzynski, K., V. Martinez, and R. S. Gupta. 1992. Immunocytochemicallocalization of heat-shock protein 60-related protein in beta-cell secretorygranules and its altered distribution in non-obese diabetic mice. Diabetolo-gia 35:316–324.

7. Burston, S. G., J. S. Weissman, G. W. Farr, W. A. Fenton, and A. L. Horwich.1996. Release of both native and non-native proteins from a cis-only GroELternary complex. Nature 383:96–99.

8. Clark, A. C., E. Hugo, and C. Frieden. 1996. Determination of regions in thedihydrofolate reductase structure that interact with the molecular chapero-nin GroEL. Biochemistry 35:5893–5901.

9. de Graeff-Meeder, E. R., G. T. Rijkers, M. M. Voorhorst-Ogink, W. Kuis, R.

van der Zee, W. van Eden, and B. J. M. Zegers. 1993. Antibodies to humanHsp60 in patients with juvenile chronic arthritis, diabetes mellitus, and cysticfibrosis. Pediatr. Res. 34:424–428.

10. Depierre, J. W., and G. Dallner. 1975. Structural aspects of the membrane ofthe endoplasmic reticulum. Biochim. Biophys. Acta 415:411–472.

11. Dignam, J. D. 1990. Preparation of extracts from higher eukaryotes. Meth-ods Enzymol. 182:194–203.

12. D’Souza, S. D., J. P. Antel, and M. S. Freedman. 1994. Cytokine induction ofheat shock protein expression in human oligodendrocytes: an interleukin-1mediated mechanism. J. Neuroimmunol. 50:17–24.

13. Ernani, F. P., and J. M. Teale. 1993. Release of stress proteins from Meso-cestoides corti is a brefeldin A-inhibitable process: evidence for active exportof stress proteins. Infect. Immun. 61:2596–2601.

14. Fayet, O., T. Ziegelhoffer, and C. Georgopoulos. 1989. The groES and groELheat shock gene products of Escherichia coli are essential for bacterialgrowth at all temperatures. J. Bacteriol. 171:1379–1385.

15. Fenton, W. A., and A. L. Horwich. 1997. GroEL-mediated protein folding.Protein Sci. 6:743–760.

16. Fenton, W. A., Y. Kashl, K. Furtak, and A. L. Horwich. 1994. Residues inchaperonin GroEL required for polypeptide binding and release. Nature371:614–619.

17. Fisch, P., M. Malkovsky, S. Kovats, E. Strum, E. Braakman, and P. Sondel.1990. Recognition by human Vg9/Vd2 T cells of a GroEL homolog on DaudiBurkitt’s lymphoma cells. Science 250:1269–1273.

18. Hiromatsu, K., Y. Yoshikai, G. Matsuzaki, S. Ohga, K. Muramori, K. Ma-tsumoto, J. A. Bluestone, K. Nomoto. 1992. A protective role of gd T cells inprimary infection with Listeria monocytogenes in mice. J. Exp. Med. 175:49–56.

19. Holoshitz, J., F. Koning, J. E. Coligan, J. de Bruyn, and S. Strober. 1989.Isolation of CD42CD82 mycobacteria-reactive T lymphocyte clones fromrheumatoid arthritis synovial fluid. Nature 339:226–229.

20. Huang, L., G. Soldevilla, M. Leeker, and R. Flavell. 1994. The liver elimi-nates T cells undergoing antigen-triggered apoptosis in vivo. Immunity1:741–749.

21. Huang, L., K. Sye, and I. N. Crispe. 1994. Proliferation and apoptosis ofB2201CD42 CD82TCRabintermediate T cells in the liver of normal adultmice: implication for lpr pathogenesis. Int. Immunol. 6:533–540.

22. Hubbard, A. L., D. A. Wall, and A. Ma. 1983. Isolation of rat hepatocyteplasma membranes. I. Presence of the three major domains. J. Cell Biol.96:217–229.

23. Itoh, H., R. Kobayashi, H. Wakui, A. Komatsuda, H. Ohtani, A. B. Miura, M.Otaka, O. Masamune, H. Andoh, K. Koyama, Y. Sato, and Y. Tashima. 1995.Mammalian 60-kDa stress protein (chaperonin homolog): identification, bio-chemical properties, and localization. J. Biol. Chem. 270:13429–13435.

24. Jarjour, W., L. A. Mizzen, W. J. Welch, S. Denning, M. Shaw, T. Mimura,B. F. Haynes, and J. B. Winfield. 1990. Constitutive expression of a groEL-related protein on the surface of human g/d cells. J. Exp. Med. 172:1857–1860.

25. Jett, M., T. M. Seed, and G. A. Jamieson. 1977. Isolation and characteriza-tion of plasma membranes and intact nuclei from lymphoid cells. J. Biol.Chem. 252:2134–2142.

26. Jones, D. B., A. F. W. Coulson, and G. W. Duff. 1993. Sequence homologiesbetween hsp60 and autoantigens. Immunol. Today 14:115–118.

27. Kaufmann, S. H. E. 1991. Heat-shock proteins and pathogenesis of bacterialinfections. Springer Semin. Immunopathol. 13:25–36.

28. Kaufmann, S. H. E. 1990. Heat-shock proteins and the immune response.Immunol. Today. 11:129–136.

29. Khan, I. U., R. Wallin, R. S. Gupta, and G. M. Kammer. 1998. Protein kinaseA-catalyzed phosphorylation of heat shock protein 60 chaperone regulatesits attachment to histone 2B in the T lymphocyte plasma membrane. Proc.Natl. Acad. Sci. USA 95:10425–10430.

30. Mayhew, M., A. C. R. da Silva, J. Martin, H. Erdjument-Bromage, P.Tempst, and F. U. Hartl. 1996. Protein folding in the central cavity of theGroEL-GroES chaperonin complex. Nature 379:420–426.

31. McVay, L. D., B. Li, R. Biancaniello, M. A. Creighton, D. Bachwich, G.Lichtenstein, J. Rombeau, and S. R. Carding. 1997. Changes in humanmucosal gd T cell repertoire and function associated with the disease processin inflammatory bowel disease. Mol. Med. 3:183–203.

32. Morimoto, R. I. 1991. Heat shock: the role of transient inducible responsesin cell damage, transformation and differentiation. Cancer Cells 3:297–301.

33. Morimoto, R. I., A. Tissieres, and C. Georgopoulos. 1989. Introduction tostress proteins, p. 1–30. In R. I. Morimoto and A. Tissieres (ed.), The role ofheat shock and stress response in biology and human disease. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.

34. O’Brien, R. L., Y.-X. Fu, R. Cranfill, A. Dallas, C. Ellis, C. Reardon, J. Lang,S. R. Carding, R. Kubo, and W. Born. 1992. heat shock protein Hsp60-reactive gd cells; a large, diversified T-lymphocyte subset with highly focusedspecificity. Proc. Natl. Acad. Sci. USA 89:4348–4352.

35. O’Brien, R. L., M. P. Happ, A. Dallas, R. Cranfill, L. Hall, J. Lang, Y.-X. Fu,R. Kubo, and W. Born. 1991. Recognition of a single hsp-60 epitope by anentire subset of gd T lymphocytes. Immunol. Rev. 121:155–169.



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

36. Ozols, J. 1990. Preparation of membrane fractions. Methods Enzymol. 182:225–235.

36a.Nosheny, R., P. Egan, and S. R. Carding. Unpublished observations.37. Peetermans, W. E., G. R. D’Haens, J. L. Ceuppens, P. Rutgeerts, and K.

Geboes. 1995. Mucosal expression by B7-positive cells of the 60-kilodaltonheat-shock protein in inflammatory bowel disease. Gastroenterology 108:75–82.

38. Perraut, R., A. R. Lussow, S. Gavoille, O. Garraud, H. Matile, C. Tougne, J.van Embden, R. van der Zee, P.-H. Lambert, J. Gysin, and G. Del Giudice.1993. Successful primate immunization with peptides conjugated to purifiedprotein derivative or mycobacterial heat shock proteins in the absence ofadjuvants. Clin. Exp. Immunol. 93:382–386.

39. Peterman, G. M., C. Spencer, A. I. Sperling, and J. A. Bluestone. 1993. Roleof gd T cells in murine collagen-induced arthritis. J. Immunol. 151:6546–6558.

40. Poccia, F., P. Piselli, S. Vendetti, S. Bach, A. Amendola, R. Placido, and V.Colizzi. 1996. Heat-shock protein expression on the membrane of T cellsundergoing apoptosis. Immunology 88:6–12.

41. Portnoy, D. A., R. K. Tweten, M. Kehoe, and J. Bielecki. 1992. Capacity oflisteriolysin O, streptolysin O, and perfringolysin O to mediate growth ofBacillus subtilis within mammalian cells. Infect. Immun. 60:2710–2717.

42. Roark, C. E., M. K. Vollmer, R. L. Cranfill, S. R. Carding, W. K. Born, andR. L. O’Brien. 1993. Liver gd T cells: TCR junctions reveal differences in

heat shock protein-60-reactive cells in liver and spleen. J. Immunol. 150:4867–4875.

43. Roseman, A. M., S. Chen, H. White, K. Braig, and H. R. Saibil. 1996. Thechaperonin ATPase cycle: mechanism of allosteric switching and movementsof substrate-binding domains in GroEL. Cell 87:241–251.

44. Selmaj, K., C. F. Brosnan, and C. S. Raine. 1991. Colocalization of lympho-cytes bearing gd T-cell receptor and heat shock protein hsp651 oligoden-drocytes in multiple sclerosis. Proc. Natl. Acad. Sci. USA 88:6452–6456.

45. Selmaj, K., C. F. Brosnan, and C. S. Raine. 1992. Expression of heat shockprotein-65 by oligodendricytes in vivo and in vitro: implications for multiplesclerosis. Neurology 42:795–802.

46. Storrie, B., and E. A. Madden. 1990. Isolation of subcellular organelles.Methods Enzymol. 182:203–225.

47. van Eden, W., J. E. R. Thole, R. van der Zee, A. Noordzij, J. D. A. vanEmbden, J. J. Henson, and I. R. Cohen. 1988. Cloning of the mycobacterialepitope recognized by T lymphocytes in adjuvant arthritis. Nature 331:171–173.

48. Viitanen, P. V., A. A. Gatenby, and G. H. Lorimer. 1992. Purified chaperonin60 (groEL) interacts with the nonnative states of a multitude of Escherichiacoli proteins. Protein Sci. 1:363–369.

49. Vincent, M. S., K. Roessner, D. Lynch, D. Wilson, S. M. Cooper, J. Tschopp,L. H. Sigal, and R. C. Budd. 1996. Apoptosis of Fas-high CD41 synovial Tells by Borrelia-reactive Fas-ligand-high gd T cells in Lyme arthritis. J. Exp.Med. 184:2109–2117.

Editor: S. H. E. Kaufmann



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

plasma membrane expression of heat shock protein 60 in ... · strepavidin-fitc, -pe, and -red670...

Documents